In this competitive renewal of a longstanding Program Project grant, multidisciplinary approaches are employed to address a central theme of gene regulation in developing red blood cells. An underlying premise in our work is that progress in the fundamental biology in the erythroid system will provide new knowledge that will have a substantial impact on the potential management of hematopoietic disorders, specifically the hemoglobinopathies and thalassemias. In aggregate, these disorders are a major health burden to society in terms of morbidity and mortality. The Program is comprised of molecular and genetic strategies that utilize the principal vertebrate model organisms-mice and zebrafish- as well as integrative genomics applied to humans. Extensive interactions between the Project Leaders will extend the capabilities of each participating laboratory beyond its own expertise and provide for synergy in execution of the proposed research. In the first Project , the transcriptional network in erythroid cells will be delineated through genome wide Chip-Chip analyses of target genes for the major transcriptional factors, GATA-1, Stat5, and FOG. In parallel, transduction pathways activated following adhesion of erythroid progenitors to fribronectin will be dissected in an effort to integrate signals with transcriptional effects. In the second Project, new genetic approaches will be undertaken to identify transcription factors controlling the fetal-to-adult human hemoglobin switch. Integrative genomic analysis of the F-cell locus previiously mapped to Xp22 will be employed to identify the relevant gene and its sequence variation in the Jamaican population. In addition, the hypothesis that Ras activity influences HbF expression will be tested, and unbiased genetic screens for new effectors of hemoglobin switching will be performed. The third Project focuses on the role of a newly identified essential erythroid nuclear factor, known as Tifly, which was first isolated from studies of mutant zebrafish. Through the use of biochemical and genetic approaches, the role of this nuclear factor will be dissected and related to the key steps in erythroid development. The fourth Project focuses on a newly identified potential regulator of adult erythroid development, zfp148. Preliminary data provide strong evidence that this protein cooperates in some unknown manner with GATA-1 and FOG-1 (and possibly other erythroid transcription factors) and may also exert stage-specificity in its action. In addition to identifying its roles in erythroid development, Cantor and Orkin will jointly investigate its potential function in hemoglobin switching. In the fifth Project, the mechanism by which the newly discovered mitochrondrial iron transporter (Frascati, mitoferrin) functions will be pursued, as well as the transcriptional effects of iron depletion on erythroid gene expression. Each of the Projects of this Program addresses new opportunities in the biology of erythroid cell development.